Elastin prevents occlusion of body vessels by vascular smooth muscle cells

Abstract

The present invention provides methods and compositions for promoting elastin signaling in smooth muscle cells including vascular smooth muscle cell. The present invention further provides methods of identifying additional agents that promote elasfin signaling.

Description

RELATED APPLICATIONS [0001] This application claims priority to U.S. provisional application 60 / 368,084 filed Mar. 27, 2002, the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disorder that causes hemodynamically significant narrowing of arteries. Although the aorta is most frequently diseased, any artery can be affected, including the pulmonary, carotid and coronary arteries. The onset and severity of vascular disease in SVAS is variable. In general a pediatric population without common risk factors such as smoking, high serum cholesterol, and high blood pressure is most affected. The pathology of SVAS arteries includes hypertrophy and hyperplasia of vascular smooth muscle cells, disrupted and disorganized elastic fibers, abundant deposition of matrix components like collagen, and inflammatory cells. These findings are commonly observed not only in ...

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Benefits of technology

[0084]FIG. 12: Elastin peptides cause a threefold increase in the F:G actin ratio. (A) Western blot analysis of − / − vascular smooth muscle cells demonstrates that treatment with either tropoelastin (SEQ ID NO: 2) or with an elastin peptide (SEQ ID NO: 3) does not alter the protein levels of either α smooth muscle actin or vinculin. (B) The effect of tropoelastin and elastin peptides on vascular smooth muscle cells does not require translation. Pretreatment of − / − vascular smooth muscle cells with either actinomycin D or cyclohexamide does not block the effects of tropoelastin or elastin peptides. (C-D) A 3-fold increase in the F:G actin ratio was observed in cells treated with either tropoelastin (SEQ ID NO: 2) or elastin peptide (SEQ ID NO: 3), but not in untreated cells or cells treated with control peptide (SEQ ID NO: 4). Phalloidin, an F-actin stabilizing agent, served as a positive control and cytochalasin D, as F-actin destabilizing agent, served as a negative control.

[0085]FIG. 13: Elastin peptides mediate actin polymerization via the small GTPase, RhoA. (A) Following 3 hours of treatment with either tropoelastin (SEQ ID NO: 2) or elastin peptide (SEQ ID NO: 3), 70-80% of elastin − / − vascular smooth muscle cells exhibited actin stress fibers. This effect was significantly decreased if cells were pretreated with either C-3 coenzyme or Y-27632. C-3 coenzyme is a Rho GTPase inhibitor and Y-27632 is a Rho kinase inhibitor. (B) Treatment with elastin peptide or tropoelastin activates RhoA by facilitating the exchange of GDP for GTP. Briefly, cell lysates from treated or untreated − / − cells were immunoprecipitated using Rhotekin-coated beads which recognize GTP-bound RhoA. Western blot analysis of these lysates using an antibody against Rho demonstrates that both tropoelastin and elastin peptide result in a significant increase of GTP-bound (activated) RhoA. Rho-γGTP served as a positive control and Rho-GDP served as a negative control.

[0086]FIG. 14: Elastin peptides mediate actin polymerization via a G-protein-coupled signaling pathway. (A-B) Pretreatment of elastin − / − cells with the heterotrimeric G protein inhibitor pertussis toxin significantly decreases the effect of tropoelastin or elastin peptide on myofilament organization and on the F:G actin ratio, however pretreatment with the β protomer (inactivate form of pertussis toxin) does not have such an effect. (C-D) The effect of tropoelastin and elastin peptide on vascular smooth muscle cells are not altered by pretreatment with either EDTA or the receptor tyrosine kinase inhibitor genistein. (E) To examine the effect of elastin signaling on c AMP levels, − / − vascular smooth muscle cells were pretreated with forskolin to elevate basal cAMP levels. Subsequent treatment of these cells with tropoelastin or elastin peptide, but not with control peptide, resulted in a decrease of cAMP levels. (F) The effect of tropoelastin or elastin peptide on forskolin induced upregulation of cAMP levels is blocked by treatment with pertussis toxin but not by treatment with the α-protomer. (G) In contrast to the activation of RhoA observed when cells are treated with either tropoelastin or elastin peptide, cells pretreated with pertussis toxin are unable to activate RhoA.

[0087]FIG. 15: Elastin peptides mediate vascular smooth muscle cell chemotaxis through G-proteins. A modified Boyden-chamber assay was used to determine the total number of migrated cells in 15 randomly selected high-power microscopic fields (HPF). (A) 20 nM elastin peptide (SEQ ID NO: 3) regulated vascular smooth muscle migration in a manner comparable to both tropoelastin and PDGF. (B-C) The effect of elastin peptide was insensitive to treatment with either the integrin inhibitor EDTA or the tyrosine kinase inhibitor genistein. However, EDTA was able to block migration to the known integrin ligand collagen, and genistein was able to block migration to the receptor tyrosine kinase ligand PDGF. (D) Pertussis toxin, but not the inactive β-protomer, blocks the effects of elastin peptide on cell migration but has no impact on PDGF induced migration. (E) The Rho kinase inhibitor Y-27632 does not block the chemotactic activity of the elastin peptide.

Problems solved by technology

If untreated, SVAS and other obstructive vascular diseases can lead to heart failure, myocardial infarction, stroke and death.

Second, large rearrangements and point mutations that disrupt ELN are associated with the disease in SVAS families.

First, injury to the vessel wall incites inflammation.

Third, some of these paracrine factors induce vascular smooth muscle cells to dedifferentiate from a quiescent contractile phenotype to a proliferative non-contractile phenotype.

Though these strategies have succeeded in culture, they have not proven to be effective in animal models or clinical settings.

However, only 70% of angioplasties lead to long-term (6 months) relief of arterial obstruction.

Such restenosis is also observed in other procedures which cause injury to a body vessel wall including the placement of stents, wires, catheters, and other intraluminal devices.

Continued advances in the geometry and composition of stents have largely impacted ease of stent delivery, but have not lessened the complication of restenosis.

The long-term efficacy is currently being tested, however given the toxicity of each agent it is doubtful that they can be used in combination with one another.

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